Calmodulin effects a tight coupling of calcium and cAMP regulation of cellular processes by controlling cAMP levels and cAMP-dependent phosphorylation. It also interacts with the regulatory subunit of cAMP dependent protein kinase and activates a calcium regulated protein phosphatase, calcineurin. The interaction of calmodulin with calcium and its target proteins is being studied in order to understand the mechanism of the regulation of cellular processes by calcium and cAMP. Using large calmodulin fragments obtained in highly purified states by HPLC, we have identified the two high affinity calcium-binding sites as sites III and IV. Calmodulin fragment 78-148 (sites III and IV) interacts with two different enzymes and with anticalmodulin drugs. The amino-terminal fragment 1-77 also interacts with anticalmodulin drugs and is required for activation of one enzyme studied but not the other. Thus, calmodulin contains at least two drug-interacting domains and different domains are required for activation of different enzymes. A covalent adduct of calmodulin with one mol of norchlorpromazine (CAPP 1-calmodulin) has been prepared. CAPP 1-calmodulin binds to calmodulin-dependent enzymes with high affinity (Ki = 10 nM - 1 nM) but has lost the ability to activate cAMP phosphodiesterase and myosin kinase and is therefore a specific and potent antagonist of calmodulin stimulation of these enzymes. It partially stimulates the phosphatase activity of calcineurin acting as a partial agonist in this case. It fully activates the calmodulin-dependent multifunctional kinase and phosphorylase kinase and does not inhibit protein kinase-C. CAPP 1-calmodulin should be a useful tool to dissect the role of calmodulin and the involvement of distinct calmodulin-regulated enzymes in cellular regulation.